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Background: Colorectal cancer (CRC) is increasing in low- and middle-income countries, likely due to changing lifestyle habits, including diet. We aimed to investigate the relationship between dietary betaine, choline, and choline-containing compounds and CRC risk. Methods: We analyzed data from a case–control study, including 865 CRC cases and 3206 controls from Iran. Detailed information was collected by trained interviewers using validated questionnaires. The intake of free choline, phosphocholine (Pcho), glycerophosphocholine (GPC), phosphatidylcholine (PtdCho), and sphingomyelin (SM), as well as of betaine was estimated from food frequency questionnaires and categorized into quartiles. The odds ratios (OR) and 95% confidence intervals (CI) of CRC for choline and betaine quartiles were calculated using multivariate logistic regression by adjusting for potential confounders. Results: We observed excess risk of CRC in the highest versus lowest intake of total choline (OR = 1.23, 95% CI 1.13, 1.33), GPC (OR = 1.13, 95% CI 1.00, 1.27), and SM (OR = 1.14, 95% CI 1.01, 1.28). The intake of betaine exerted an inverse association with CRC risk (OR = 0.91, 95% CI 0.83, 0.99). There was no association between free choline, Pcho, PtdCho, and CRC. Analyses stratified by gender showed an elevated OR of CRC in men for SM intake OR = 1.20, 95% CI 1.03, 1.40) and a significantly decreased CRC risk in women for betaine intake (OR = 0.84, 95% CI 0.73, 0.97). Conclusion: Dietary modifications leading to an increase in betaine sources and managing the use of animal products as references for SM or other choline types might contribute to decreasing the risk of CRC. publishedVersion

Antimicrobial peptides (AMPs), which typically disrupt the bacterial wall prompting leakage or lysis of the cell, form a growing contingent in the arsenal against antibiotic resistant bacteria. The effectiveness of AMPs is, however, hampered by their low solubility, general chemical and physical instability, and short half-life in vivo. Lipid nanocarriers such as cubosomes are effective at encapsulating and protecting proteins while simultaneously showing promise in delivery applications. Here, the efficacy of cubosome mediated delivery of AMPs is evaluated by the in-situ surface characterization of model membranes with varying composition. The cubosomes were observed to initially fuse with the membranes, with subsequent membrane disruption observed after approximately 20 - 60 min. The time for the disruption was sensitive to the charge of the cubosome as well as the composition of the bilayer. More physiologically relevant bilayers including lipids with phospho-(1'-rac-glycerol) (PG) or phosphoethanolamine (PE) headgroups were more vulnerable than those of neat phosphocholine (PC). Notably, disruption to the bilayer occurred an order of magnitude faster for encapsulated AMP compared to free AMP.

The tissue metabolomic characteristics associated with endometrial cancer (EC) at different grades were studied using high resolution (400 MHz) magic angle spinning (HR-MAS) proton spectroscopy. The metabolic profiles were obtained from 64 patients (14 with grade 1 (G1), 33 with grade 2 (G2) and 17 with grade 3 (G3) tumors) and compared with the profile acquired from 10 patients with the benign disorders. OPLS-DA revealed increased valine, isoleucine, leucine, hypotaurine, serine, lysine, ethanolamine, choline and decreased creatine, creatinine, glutathione, ascorbate, glutamate, phosphoethanolamine and scyllo-inositol in all EC grades in reference to the non-transformed tissue. The increased levels of taurine was additionally detected in the G1 and G2 tumors in comparison to the control tissue, while the elevated glycine, N-acetyl compound and lactate—in the G1 and G3 tumors. The metabolic features typical for the G1 tumors are the increased dimethyl sulfone, phosphocholine, and decreased glycerophosphocholine and glutamine levels, while the decreased myo-inositol level is characteristic for the G2 and G3 tumors. The elevated 3-hydroxybutyrate, alanine and betaine levels were observed in the G3 tumors. The differences between the grade G1 and G3 malignances were mainly related to the perturbations of phosphoethanolamine and phosphocholine biosynthesis, inositol, betaine, serine and glycine metabolism. The statistical significance of the OPLS-DA modeling was also verified by an univariate analysis. HR-MAS NMR based metabolomics provides an useful insight into the metabolic reprogramming in endometrial cancer.

Self-assembly of biomembranes results from the intricate interactions between water and the lipids’ hydrophilic head groups. Therefore, the lipid–water interplay strongly contributes to modulating membrane architecture, lipid diffusion, and chemical activity. Here, we introduce a new method of obtaining dehydrated, phase-separated, supported lipid bilayers (SLBs) solely by controlling the decrease of their environment’s relative humidity. This facilitates the study of the structure and dynamics of SLBs over a wide range of hydration states. We show that the lipid domain structure of phase-separated SLBs is largely insensitive to the presence of the hydration layer. In stark contrast, lipid mobility is drastically affected by dehydration, showing a 6-fold decrease in lateral diffusion. At the same time, the diffusion activation energy increases approximately 2-fold for the dehydrated membrane. The obtained results, correlated with the hydration structure of a lipid molecule, revealed that about six to seven water molecules directly hydrating the phosphocholine moiety play a pivotal role in modulating lipid diffusion. These findings could provide deeper insights into the fundamental reactions where local dehydration occurs, for instance during cell–cell fusion, and help us better understand the survivability of anhydrobiotic organisms. Finally, the strong dependence of lipid mobility on the number of hydrating water molecules opens up an application potential for SLBs as very precise, nanoscale hydration sensors.

The objective of the present study was to investigate the different plasma metabolites between anestrus and estrus postpartum dairy cows and to provide a theoretical basis for prevention of anestrus in dairy farm cows. In the experiment, one hundred and sixty-seven Holstein dairy cows were selected with similar age and parity. According to the concentration of β-hydroxybutyric acid, non-esterified fatty acids and glucose in plasma during 14 to 21 days in milk, all dairy cows were determined as having a status of energy balance. According to the results of clinical symptom, rectal and B ultrasound examination at 60 to 90 days postpartum, these cows were divided into twenty estrus and twenty-four anestrus group, other dairy cows were removed. 1H nuclear magnetic resonance technology was utilized to detect the plasma metabolites changes and screen different plasma metabolites between anestrus and estrus cows. Ten different metabolites including alanine, glutamic acid, asparagine, creatine, choline, phosphocholine, glycerophosphocholine, low-density lipoprotein, and very-low-density lipoprotein were significantly decreased in anestrous cows compared with estrous cows. Metabolic pathway analyses indicated that differential metabolites were primarily involved in amino acid and glycerophospholipid metabolism. These metabolites and their enrichment pathways indicate that reduced steroid hormone synthesis precursors result in lower levels of estradiol and progesterone and cause anestrus in negative energy balance. These data provide a better understanding of the changes that may affect estrus of postpartum dairy cows at NEB status and lay the ground for further research.

Mesocotyl elongation of rice is crucial for seedlings pushing out of deep soil. The underlying mechanisms of phospholipid signaling in mesocotyl growth of rice are elusive. Here we report that the rice non-specific phospholipase C6 (OsNPC6) is involved in mesocotyl elongation. Our results indicated that all five OsNPCs (OsNPC1, OsNPC2, OsNPC3, OsNPC4 and OsNPC6) hydrolyzed the substrate phosphatidylcholine to phosphocholine (PCho), and all of them showed plasma membrane localization. Overexpression (OE) of OsNPC6 produced plants with shorter mesocotyls compared to those of Nipponbare and npc6 mutants. Although the mesocotyl growth of npc6 mutants was not much affected without gibberellic acid (GA)3, it was obviously elongated by treatment with GA. Upon GA3 treatment, SLENDER RICE1 (SLR1), the DELLA protein of GA signaling, was drastically increased in OE plants; by contrast, the level of SLR1 was found decreased in npc6 mutants. The GA-enhanced mesocotyl elongation and the GA-impaired SLR1 level in npc6 mutants were attenuated by the supplementation of PCho. Further analysis indicated that the GA-induced expression of phospho-base N-methyltransferase 1 in npc6 mutants was significantly weakened by the addition of PCho. In summary, our results suggest that OsNPC6 is involved in mesocotyl development via modulation of PCho in rice.

The current approaches remain insufficient for measuring chicken egg spoilage or present analytical limitations. This study aimed to complement the existing analyses and identify novel markers using liquid chromatography-high resolution mass spectrometry-based foodomics strategies. In the discovery set, comparative untargeted metabolomics was utilized to identify marker candidates in microbially inoculated chicken eggs. Markers were annotated by spectral matching with authentic standards, experimental libraries, or in silico fragmentation. In the validation set, targeted metabolomics was employed to verify the markers in stored chicken eggs from five farms. Statistical differences at a p-value < 0.001 revealed increases in lactic and 3-hydroxybutyric acids and decreases in phosphocholine, LPE(O-18:1), LPC(16:0), and LPC(18:0) in stored eggs. Receiver operating characteristic curve analysis of the six combined markers yielded an AUC of 0.956 and a sensitivity and specificity of ∼90%. Four phospholipids were highlighted as a novel class of spoilage markers. Our findings may contribute to further industrial implementation, benefiting the quality assurance and food safety of poultry egg production.

INTRODUCTION: Altered lipid metabolism and subsequent changes in cellular lipid composition have been observed in prostate cancer cells, are associated with poor clinical outcome, and are promising targets for metabolic therapies. This study reports for the first time on the synthesis of a phospholipid radiotracer based on the phospholipid 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine (PC44:12) to allow tracking of polyunsaturated lipid tumor uptake via PET imaging. This tracer may aid in the development of strategies to modulate response to therapies targeting lipid metabolism in prostate cancer. METHODS: Lipidomics analysis of prostate tumor explants and LNCaP tumor cells were used to identify PC44:12 as a potential phospholipid candidate for radiotracer development. Synthesis of phosphocholine precursor and non-radioactive standard were optimised using click chemistry. The biodistribution of a fluorine-18 labeled analogue, N-{[4-(2-[18F]fluoroethyl)-2,3,4-triazol-1-yl]methyl}-1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine ([18F]2) was determined in LNCaP prostate tumor-bearing NOD SCID gamma mice by ex vivo biodistribution and PET imaging studies and compared to biodistribution of [18F]fluoromethylcholine. RESULTS: [18F]2 was produced with a decay-corrected yield of 17.8 ± 3.7% and an average radiochemical purity of 97.00 ± 0.89% (n = 6). Molar activity was 85.1 ± 3.45 GBq/μmol (2300 ± 93 mCi/μmol) and the total synthesis time was 2 h. Ex vivo biodistribution data demonstrated high liver uptake (41.1 ± 9.2%ID/g) and high splenic uptake (10.9 ± 9.1%ID/g) 50 min post-injection. Ex vivo biodistribution showed low absolute tumor uptake of [18F]2 (0.8 ± 0.3%ID/g). However, dynamic PET imaging demonstrated an increase over time of the relative tumor-to-muscle ratio with a peak of 2.8 ± 0.5 reached 1 h post-injection. In contrast, dynamic PET of [18F]fluoromethylcholine demonstrated no increase in tumor-to-muscle ratios due to an increase in both tumor and muscle over time. Absolute uptake of [18F]fluoromethylcholine was higher and peaked at 60 min post injection (2.25 ± 0.29%ID/g) compared to [18F]2 (1.44 ± 0.06%ID/g) during the 1 h dynamic scan period. CONCLUSIONS AND ADVANCES IN KNOWLEDGE: This study demonstrates the ability to radiolabel phospholipids and indicates the potential to monitor the in vivo distribution of phospholipids using fluorine-18 based PET. ispartof: NUCLEAR MEDICINE AND BIOLOGY vol:93 pages:37-45 ispartof: location:United States status: published

In this study, we describe the engineering of sub-100 nm nanomicelles (DTX-PC NMs) derived from phosphocholine derivative of docetaxel (DTX)-conjugated lithocholic acid (DTX-PC) and poly(ethylene glycol)-tethered lithocholic acid. Administration of DTX-PC NMs decelerate tumor progression and increase the mice survivability compared to Taxotere (DTX-TS), the FDA-approved formulation of DTX. Unlike DTX-TS, DTX-PC NMs do not cause any systemic toxicity and slow the decay rate of plasma DTX concentration in rodents and non-rodent species including non-human primates. We further demonstrate that DTX-PC NMs target demethylation of CpG islands of Sparcl1 (a tumor suppressor gene) by suppressing DNA methyltransferase activity, and increase the expression of Sparcl1 that leads to tumor regression. Therefore, this unique system has the potential to improve the quality of life in cancer patients, and can be translated as a next-generation chemotherapeutic.

To determine the effect of feeding buttermilk-derived choline metabolites on the immune system development in Sprague–Dawley rat pups. Sprague–Dawley dams were randomized to one of the three diets containing 1.7 g/kg choline: 1-Control (100% free choline (FC)), 2-Buttermilk (BM, 37% phosphatidylcholine (PC), 34% sphingomyelin (SM), 17% glycerophosphocholine (GPC), 7% FC, 5% phosphocholine), and 3-Placebo (PB, 50% PC, 25% FC, 25% GPC) until the end of the lactation period. At weaning, pups continued on the same diet as their mom. Cell phenotypes and cytokine production by mitogen-stimulated splenocytes isolated from 3- and 10-week-old pups were measured. At 3 weeks, BM-pups had a higher proportion of cytotoxic T cells (CTL; CD3 + CD8 +) while both BM- and PB-pups had an increased proportion of cells expressing CD28 + , CD86 + and CD27 + (all p > 0.05). Following ConA stimulation, splenocytes from BM- and PB-pups produced more TNF-α and IFN-γ and after LPS stimulation produced more IL-10 and TNF-α (all p > 0.05). Starting at week 6 of age, BM-pups had a higher body weight. At 10 weeks, both the BM- and PB-pups had a higher proportion of CTL expressing CD27 + . After ConA stimulation, splenocytes from BM- and PB-pups produced more IL-2, IFN-γ and IL-6 and more IL-10 after LPS stimulation (all p > 0.05). The proportion of lipid soluble forms of choline in the diet during lactation and weaning periods influence the immune system development in rat offspring.

Hybrid siloxane-phosphocholines (SiPCs) are a unique class of lipids that possess the capacity to spontaneously form unilamellar vesicles (ULVs) that are ∼100 nm in diameter upon exposure to aqueous media without the need for extrusion. These hybrid SiPCs do not negatively impact the growth of a number of bacterial strains and were subsequently explored as candidates for the delivery of a model compound, calcein. Liposomes that were formed spontaneously encapsulated larger quantities of calcein than liposomes that were formed via extrusion (both SiPCs and commercially available lipids), indicating that the extrusion process results in the loss of the material being entrapped and decreased entrapment efficiency. Although the percentage of calcein released was virtually identical across liposome samples (extruded and non-extruded), the greater entrapment efficiency of the spontaneously formed liposomes ultimately results in a greater payload delivery.

1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC) coated on the surface of superparamagnetic iron oxide nanoparticles (SPIONs) has advantages in neurotherapy and drug delivery. In this study, the surface of polyvinylpyrrolidone (PVP)-SPIONs was modified with DMPC, then PVP-SPIONs and DMPC/PVP-SPIONs were co-incubated with rat adrenal pheochromocytoma (PC-12) cells to observe the effect of DMPC on the distribution of SPIONs in cells, and further PVP-SPIONs and DMPC/PVP-SPIONs were implanted into the substantia nigra of Sprague-Dawley (SD) rats by stereotaxic injection, and the brain tissues were removed at both twenty-four hours and seven days after injection. The distribution and transport of nanoparticles in the substantia nigra in vivo were explored in these different time periods. The results show that DMPC/PVP-SPIONs were effectively distributed on the membranes of axons, as well as dendritic and myelin sheaths. The attachment of nanoparticles to bio-membranes in the brain could result from similar phospholipid structures of DMPC and the membranes. In addition, DMPC/PVP-SPIONs were transported in the brain faster than those without DMPC. In vitro experiments found that DMPC/PVP-SPIONs enter cells more easily. These characteristics of iron oxide nanoparticles that are modified by phospholipids lead to potential applications in drug delivery or activating neuron membrane channels.

Background Proton magnetic resonance spectroscopy (MRS) provides a unique opportunity for in vivo measurements of the brain's metabolic profile. Two methods of mainstream data acquisition are compared at 7 T, which provides certain advantages as well as challenges. The two representative methods have seldom been compared in terms of measured metabolite concentrations and different scan times. The current study investigated proton MRS of the posterior cingulate cortex using a semi-localized by adiabatic selective refocusing (sLASER) sequence and a short echo time (TE) stimulated echo acquisition mode (sSTEAM) sequence, and it compared their reliability and repeatability at 7 T using a 32-channel head coil. Methods Sixteen healthy subjects were prospectively enrolled and scanned twice with an off-bed interval between scans. The scan parameters for sLASER were a TR/TE of 6.5 s/32 ms and 32 and 48 averages (sLASER×32 and sLASER×48, respectively). The scan parameters for sSTEAM were a TR/TE of 4 s/5 ms and 32, 48, and 64 averages (sSTEAM4×32, sSTEAM4×48, and sSTEAM4×64, respectively) in addition to that with a TR/TE of 8 s/5 ms and 32 averages (sSTEAM8×32). Data were analyzed using LCModel. Metabolites quantified with Cramer-Rao lower bounds (CRLBs) >50% were classified as not detected, and metabolites quantified with mean or median CRLBs ≤20% were included for further analysis. The SNR, CRLBs, coefficient of variation (CV), and metabolite concentrations were statistically compared using the Shapiro-Wilk test, one-way ANOVA, or the Friedman test. Results The sLASER spectra for N-acetylaspartate + N-acetylaspartylglutamate (tNAA) and glutamate (Glu) had a comparable or higher SNR than sSTEAM spectra. Ten metabolites had lower CRLBs than prefixed thresholds: aspartate (Asp), γ-aminobutyric acid (GABA), glutamine (Gln), Glu, glutathione (GSH), myo-inositol (Ins), taurine (Tau), the total amount of phosphocholine + glycerophosphocholine (tCho), creatine + phosphocreatine (tCr), and tNAA. Performance of the two sequences was satisfactory except for GABA, for which sLASER yielded higher CRLBs (≥18%) than sSTEAM. Some significant differences in CRLBs were noted, but they were ≤2% except for GABA and Gln. Signal averaging significantly lowered CRLBs for some metabolites but only by a small amount. Measurement repeatability as indicated by median CVs was ≤10% for Gln, Glu, Ins, tCho, tCr, and tNAA in all scans, and that for Asp, GABA, GSH, and Tau was ≥10% under some scanning conditions. The CV for GABA according to sLASER was significantly higher than that according to sSTEAM, whereas the CV for Ins was higher according to sSTEAM. An increase in signal averaging contribute little to lower CVs except for Ins. Conclusions Both sequences quantified brain metabolites with a high degree of precision and repeatability. They are comparable except for GABA, for which sSTEAM would be a better choice.

To address biophysical principles and lipid interactions that underlie the properties of membrane proteins, modifications that vary the neighbors of tryptophan residues in the highly dynamic transmembrane helix of GW4,20 ALP23 (acetyl-GGAW4 A(LA)6 LAW20 AGA-amide) were examined using deuterium NMR spectroscopy. It was found that L5,19 GW4,20 ALP23, a sequence isomer of the low to moderately dynamic GW5,19 ALP23, remains highly dynamic. By contrast, a removal of W4 to produce F4,5 GW20 ALP23 restores a low level of dynamic averaging, similar to that of the F4,5 GW19 ALP23 helix. Interestingly, a high level of dynamic averaging requires the presence of both tryptophan residues W4 and W20, on opposite faces of the helix, and does not depend on whether residue 5 is Leu or Ala. Aspects of helix unwinding and potential oligomerization are discussed with respect to helix dynamic averaging and the locations of particular residues at a phosphocholine membrane interface.

Stress is considered as an important risk factor in the progression and the onset of many disorders such as multiple sclerosis. However, metabolite changes as a result of demyelination under the detrimental effects of stress are not well understood. Thus, 36 female Wistar rats (i.e., groups (1) no-cuprizone (Cont), (2) no-stress + cuprizone-treated (Cup), (3) physical stress + cuprizone-treated (P-Cup), (4) psychological stress + cuprizone-treated (Psy-Cup), (5) physical stress + no-cuprizone-treated (P), (6) psychological stress + no-cuprizone-treated (Psy)) were used in this study. Following induction of repetitive stress, cuprizone treatment was carried out for 6 weeks to instigate demyelination in all groups except the control animal. Relative metabolite concentrations of the brain were investigated by single-voxel proton magnetic resonance spectroscopy (reporting N-acetyl-aspartate (NAA), glycerophosphocholine with phosphocholine (tCho) relative to total creatine (tCr)). According to 1H-MRS, rats in the Cup group indicated a reduction in NAA/ tCr (p < 0.001) as well as tCho/ tCr (p < 0.05) compared with that in the Cont group. In contrast, in both stress + cuprizone-treated groups, NAA/tCr and tCho/tCr ratios remarkably increased versus the Cup group (p < 0.001) and the Cont group (p < 0.001 for the Psy-Cup group and p < 0.05 for the P-Cup group). Both P and Psy groups revealed normal metabolite concentrations similar to the Cont group 6 weeks post stress. Seemingly, in the case of cuprizone alone, decreased level of metabolites is mainly relevant to neuronal cell impairments. Meanwhile, as a result of oxidative stress enhancement due to stress exposure, oligodendrocyte becomes the main victim indicating the increased level of metabolite ratios.

Early diagnosis of Niemann-Pick diseases (NPDs) is important for better prognosis of such diseases. N-Palmitoyl-O-phosphocholine-serine (PPCS) is a new NPD biomarker possessing high sensitivity, and with its combination with sphingosylphosphocholine (SPC) it may be possible to distinguish NPD-C from NPD-A/B. In this study, a rapid liquid chromatography/tandem mass spectrometry (LC/MS/MS) method (method 1) and a validated LC/MS/MS analysis (method 2) of PPCS and SPC were developed, and we have proposed a diagnostic screening strategy for NPDs using a combination of serum PPCS and SPC concentrations.Nexera and API 5000 were used as LC/MS/MS systems. C18 columns with lengths of 10 mm and 50 mm were used for method 1 and 2, respectively. 2H3-labeled PPCS (PPCS-2H3_ and nor-SPC were used as internal standards. Selective reaction monitoring in positive-ion mode was used for MS/MS. Run times of 1.2 min and 8 min were set for methods 1 and 2, respectively.In both methods 1 and 2, two analytes showed high linearity in the range of 1-4000 ng/mL. Method 2 provided high accuracy and precision in method validation. Serum concentrations of both analytes were significantly higher in NPD-C patients than those of healthy subjects in both methods. Serum PPCS correlated between methods 1 and 2; however, it was different in the case of SPC. The serum PPCS/SPC ratio was different in healthy subjects, NPD-C, and NPD-A/B. These results suggest that using a combination of the two LC/MS/MS analytical methods for PPCS and SPC is useful for diagnostic screening of NPDs.

Macroautophagy initiates by formation of isolation membranes, but the source of phospholipids for the membrane biogenesis remains elusive. Here, we show that autophagic membranes incorporate newly synthesized phosphatidylcholine, and that CTP:phosphocholine cytidylyltransferase β3 (CCTβ3), an isoform of the rate-limiting enzyme in the Kennedy pathway, plays an essential role. In starved mouse embryo fibroblasts, CCTβ3 is initially recruited to autophagic membranes, but upon prolonged starvation, it concentrates on lipid droplets that are generated from autophagic degradation products. Omegasomes and isolation membranes emanate from around those lipid droplets. Autophagy in prolonged starvation is suppressed by knockdown of CCTβ3 and is enhanced by its overexpression. This CCTβ3-dependent mechanism is also present in U2OS, an osteosarcoma cell line, and autophagy and cell survival in starvation are decreased by CCTβ3 depletion. The results demonstrate that phosphatidylcholine synthesis through CCTβ3 activation on lipid droplets is crucial for sustaining autophagy and long-term cell survival., The source of phospholipids to generate autophagosomal membranes, particularly after prolonged starvation, is not well characterized. Here, the authors show that CCTβ3, the rate limiting enzyme in phosphatidylcholine synthesis, is activated on lipid droplets and sustains long-term autophagy.

Summary Background & aims Insulin resistance (IR) is one of the main risk factor for type 2 diabetes mellitus (T2DM). Nevertheless, its underlying pathophysiology is not completely established because IR is triggered by a complex interconnection of numerous factors impairing metabolism, promoting metabolome changes. Methods We used a metabolomics approach to identify plasma and faecal metabolites related to IR and obesity. We explored a cohort of 44 subjects at baseline, with 30 of them followed two years thereafter in a longitudinal study after an hypocaloric diet in the obese subjects. Results In all individuals as a whole, 11 plasma metabolites positively associated with BMI (acetoacetate, creatinine, glycerol, glycerol of lipids, VLDL, fatty esters, myo-inositol, phenylalanine, threonine, tyrosine and valine) and one negatively (phosphocholine), with similar associations at baseline and follow-up. Four of these metabolites (myo-inositol, valine, acetoacetate and phosphocholine) remained significant within obese and non-obese groups. Thirteen faecal metabolites positively associated with BMI at baseline and one negatively (glutamine). However, these correlations did not remain significant at follow-up. The correlations were not always consistent at baseline and at follow-up and the metabolites that showed significant correlations were different for the obese group compared with the control group. The percent change in plasma Δethanolamine, Δglucose, Δuracil and Δhypoxanthine were positively associated with ΔBMI. The percent change in plasma Δphosphocholine and of faecal Δhydroxyphenylacetate, and Δ2-hydroxyphenylacetate were associated with ΔHOMA-IR in those patients that lost weight. Faecal branched chain amino acids (BCAAs) in faeces were associated with IR, following a similar pattern to that described for plasma BCAAs. Choline derivates had an opposite behaviour. Conclusions The integration of plasma and faecal metabolites represents a valuable fingerprint that could help in the identification of patients at risk for IR and in the design of novel therapeutic strategies to prevent IR and the development of overt T2DM in the context of obesity. The results are coherent with diet having a much greater impact on faecal metabolomic profile than on plasma metabolome.

Proton nuclear spins of dodecyl phosphocholine molecules below the critical micelle concentration are hyperpolarized by using dissolution dynamic nuclear polarization (D-DNP). NMR signal enhancements of 1210±400 and 1610±550 are obtained at 9.4 T, for choline methyls in the head group of the lipid and for the tail-end methyl group, respectively. This polarization is transferred to the unfolded protein through the nuclear Overhauser effect, after dilution to a final denaturant concentration of 0.8 M urea. As a result, the amide and aromatic side-chain signals of the protein are increased up to sixfold. Selective inversion pulses applied either on the head-group or tail-group of the lipid are used to identify the source of the transferred polarization. The normalized cross-relaxation rates of σN,tail =-1.8±0.1 s-1 M-1 and σN,head =-0.5±0.3 s-1 M-1 are obtained, showing a larger polarization transfer from the tail groups. These cross-relaxation rates are determined at a low urea concentration, which constitutes refolding conditions for the protein. The sensitivity enhancement by D-DNP permits to access these conditions with a measurement time on the order of seconds, and may further open the possibility to investigate structural changes in membrane proteins during folding.

C-reactive protein (CRP) can increase up to 1000-fold in blood and form complexes with very low density lipoproteins (VLDL). These complexes are associated with worse outcomes for septic patients, and this suggests a potential pathological role in sepsis. Complex formation is heightened when CRP is over 200 mg/l and levels are associated with the severity of sepsis and blood bacterial culture positivity. Using a mouse bacteremia model, blood bacterial clearance can be delayed by i.v. injection of CRP–VLDL complexes. Complexes are more efficiently taken up by activated U937 cells in vitro and Kupffer cells in vivo than VLDL alone. Both in vitro–generated and naturally occurring CRP–VLDL complexes reduce phagocytosis of bacteria by activated U937 cells. Fcγ and scavenger receptors are involved and a competitive mechanism for clearance of CRP–VLDL complexes and bacteria is demonstrated. Interaction of phosphocholine groups on VLDL with CRP is the major driver for complex formation and phosphocholine can disrupt the complexes to reverse their inhibitory effects on phagocytosis and bacterial clearance. Increased formation of CRP–VLDL complexes is therefore harmful and could be a novel target for therapy in sepsis.

CTP:phosphocholine cytidylyltransferase-alpha (CCTα) and CCTβ catalyze the rate-limiting step in phosphatidylcholine (PC) biosynthesis. CCTα is activated by association of its α-helical M-domain with nuclear membranes, which is negatively regulated by phosphorylation of the adjacent P-domain. To understand how phosphorylation regulates CCT activity, we developed phosphosite-specific antibodies for pS319 and pY359+pS362 at the N- and C-termini of the P-domain, respectively. Oleate treatment of cultured cells triggered CCTα translocation to the nuclear envelope (NE) and nuclear lipid droplets (nLDs) and rapid dephosphorylation of pS319. Removal of oleate led to dissociation of CCTα from the NE and increased phosphorylation of S319. Choline depletion of cells also caused CCTα translocation to the NE and S319 dephosphorylation. In contrast, Y359 and S362 were constitutively phosphorylated during oleate addition and removal, and CCTα-pY359+pS362 translocated to the NE and nLDs of oleate-treated cells. Mutagenesis revealed that phosphorylation of S319 is regulated independently of Y359+S362, and that CCTα-S315D+S319D was defective in localization to the NE. We conclude that the P-domain undergoes negative charge polarization due to dephosphorylation of S319 and possibly other proline-directed sites and retention of Y359 and S362 phosphorylation, and that dephosphorylation of S319 and S315 is involved in CCTα recruitment to nuclear membranes.

Background Prostate cancer (PC) can display very heterogeneous phenotypes ranging from indolent asymptomatic to aggressive lethal forms. Understanding how these PC subtypes vary in their striving for energy and anabolic molecules is of fundamental importance for developing more effective therapies and diagnostics. Here, we carried out an extensive analysis of prostate tissue samples to reveal metabolic alterations during PC development and disease progression and furthermore between TMPRSS2-ERG rearrangement-positive and -negative PC subclasses. Methods Comprehensive metabolomics analysis of prostate tissue samples was performed by non-destructive high-resolution magic angle spinning nuclear magnetic resonance (1H HR MAS NMR). Subsequently, samples underwent moderate extraction, leaving tissue morphology intact for histopathological characterization. Metabolites in tissue extracts were identified by 1H/31P NMR and liquid chromatography-mass spectrometry (LC-MS). These metabolomics profiles were analyzed by chemometric tools and the outcome was further validated using proteomic data from a separate sample cohort. Results The obtained metabolite patterns significantly differed between PC and benign tissue and between samples with high and low Gleason score (GS). Five key metabolites (phosphocholine, glutamate, hypoxanthine, arginine and α-glucose) were identified, who were sufficient to differentiate between cancer and benign tissue and between high to low GS. In ERG-positive PC, the analysis revealed several acylcarnitines among the increased metabolites together with decreased levels of proteins involved in β-oxidation; indicating decreased acyl-CoAs oxidation in ERG-positive tumors. The ERG-positive group also showed increased levels of metabolites and proteins involved in purine catabolism; a potential sign of increased DNA damage and oxidative stress. Conclusions Our comprehensive metabolomic analysis strongly indicates that ERG-positive PC and ERG-negative PC should be considered as different subtypes of PC; a fact requiring different, sub-type specific treatment strategies for affected patients.

Background Choline is an essential nutrient for brain growth and other processes in the developing neonate. The impact of a maternal plant-based diet on the choline composition of breast milk is unknown. Objective We assessed the water-soluble choline content of milk from lactating women in the United States following 3 dietary patterns: vegan, vegetarian, and nonvegetarian. Methods We conducted a cross-sectional study of 74 healthy lactating women who provided a single breast-milk sample using a standardized collection protocol. Participants completed a food-frequency screener and were classified as follows: nonvegetarians (NONVEG) consumed meat; vegetarians (VEGT) consumed milk, dairy, and/or fish; and vegans (VEGAN) consumed animal products less than monthly. Primary outcomes measured were the concentration (in milligrams per liter) and distribution (percentage) of choline from the following water-soluble forms: free choline, phosphocholine (PCho), and glycerophosphocholine (GPC). Differences between diet groups were evaluated with ANOVA. Results There was a wide range in breast-milk total water-soluble choline (4-301 mg/L), with no significant difference (P > 0.05) by maternal diet pattern. There were differences in choline forms, with VEGAN having a greater mean ± SD concentration and distribution of choline derived from GPC (62.7 ± 25.3 mg/L) than VEGT (47.7 ± 21.2 mg/L) and NONVEG (42.4 ± 14.9 mg/L) (P = 0.0052). There was a lower mean ± SD percentage of choline from PCho (P = 0.0106) in VEGAN (32.5% ± 18.3%) than in VEGT (46.1% ± 18.3%) and NONVEG (44.8% ± 15.7%). Lactation stage and maternal BMI were significantly associated with some choline forms. Conclusions There was a wide range of water-soluble choline concentrations in the milk of healthy lactating women following vegan, vegetarian, and nonvegetarian diets, with no observed difference in total water-soluble choline concentration by maternal diet. This suggests that maternal plant-based diet by itself is not a risk factor for low breast-milk choline.

BackgroundBacterial toxins disrupt plasma membrane integrity with multitudinous effects on host cells. The secreted pore-forming toxin listeriolysin O (LLO) of the intracellular pathogen Listeria monocytogenes promotes egress of the bacteria from vacuolar compartments into the host cytosol often without overt destruction of the infected cell. Intracellular LLO activity is tightly controlled by host factors including compartmental pH, redox, proteolytic, and proteostatic factors, and inhibited by cholesterol.MethodsCombining infection studies of L. monocytogenes wild type and isogenic mutants together with biochemical studies with purified phospholipases, we investigate the effect of their enzymatic activities on LLO.ResultsHere, we show that phosphocholine (ChoP), a reaction product of the phosphatidylcholine-specific phospholipase C (PC-PLC) of L. monocytogenes, is a potent inhibitor of intra- and extracellular LLO activities. Binding of ChoP to LLO is redox-independent and leads to the inhibition of LLO-dependent induction of calcium flux, mitochondrial damage, and apoptosis. ChoP also inhibits the hemolytic activities of the related cholesterol-dependent cytolysins (CDC), pneumolysin and streptolysin.ConclusionsOur study uncovers a strategy used by L. monocytogenes to modulate cytotoxic LLO activity through the enzymatic activity of its PC-PLC. This mechanism appears to be widespread and also used by other CDC pore-forming toxin-producing bacteria.

Cucurbit[7]uril was used to form non-covalent complexes with low-molecular-weight quaternary-ammonium compounds for their indirect analysis by MALDI-MS. By shifting the ion signals to a higher and interference-free mass region, the distributions of neurine, choline, and phosphocholine in rat brain were visualized by MALDI imaging with high selectivity and good sensitivity.

Carbon nanodots (CDs) are fluorescent nanomaterials that have attracted widespread attention for biomedical applications. Since different cells possess different membrane potentials, the influence of the trans-membrane potential on the adsorption behaviour needs to be explored extensively. It was found that fewer CDs can be adsorbed by depolarized cells with decreased membrane potential, as compared to hyperpolarized cells with enhanced membrane potential that promotes the adsorption of CDs. A similar adsorption trend was observed for two cell lines, namely NIH3T3 and MCF-7. Since phosphocholine is the primary component of the cellular membrane, the adsorption behaviour of CDs is further complemented by the interactions between CDs and three kinds of liposomes with different surface charges via the thermodynamic and kinetic perspectives. The adsorption constants between negatively charged CDs and liposomes are positively related to their membrane potential, which is similar to the adsorption rate constants. It was further found that the adsorption of CDs onto liposomes was initiated by electrostatic attraction forces. The membrane integrity and fluidity of liposomes exposed to CDs have also been characterized. The membrane potential of cells is, therefore, particularly important when predicting the relative cytotoxicity of CDs and developing CDs-based nanomaterials for biomedical applications.

Knowledge of thermodynamics of lipid membrane partitioning of amphiphilic drugs as well as their binding site within the membrane are of great relevance not only for understanding the drugs' pharmacology but also for the development and optimization of more potent drugs. In this study, the interaction between two representatives of selective serotonin reuptake inhibitors, including paroxetine and sertraline, and large unilamellar vesicles (LUVs) composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) was investigated by second derivative spectrophotometry and Fourier transform infrared spectroscopy (FTIR) to determine the driving force of the drug partitioning across lipid membranes. It was found that temperature increase from 25 to 42 °C greatly enhanced the partitioning of paroxetine and sertraline into DOPC LUVs, and sertraline intercalated into the lipid vesicles to a greater extent than paroxetine in the temperature range examined. The partitioning of both drugs into DOPC LUVs was a spontaneous, endothermic and entropy-driven process. FTIR measurements suggested that sertraline could penetrate deeply into the acyl tails of DOPC LUVs as shown by the considerable shifts in the lipid's CH2 and CO stretching modes induced by the drug. Paroxetine, however, could reside closer to the head groups of the lipid since its presence caused a larger shift in the PO2− bands of DOPC LUVs. The findings reported here provide valuable insights into the influence of small molecules' chemical structure on their molecular interaction with the lipid bilayer namely their possible binding sites within the lipid bilayer and their thermodynamics profiles of partitioning, which could benefit rational drug design and drug delivery systems.

Phosphocholine molecules decorating bacterial cell wall teichoic acids and outer-membrane lipopolysaccharide have fundamental roles in adhesion to host cells, immune evasion, and persistence. Bacteria carrying the operon that performs phosphocholine decoration synthesize phosphocholine after uptake of the choline precursor by LicB, a conserved transporter among divergent species. Streptococcus pneumoniae is a prominent pathogen where phosphocholine decoration plays a fundamental role in virulence. Here, we present cryo–electron microscopy and crystal structures of S. pneumoniae LicB, revealing distinct conformational states and describing architectural and mechanistic elements essential to choline import. Together with in vitro and in vivo functional characterization, we found that LicB displays proton-coupled import activity and promiscuous selectivity involved in adaptation to choline deprivation conditions, and describe LicB inhibition by synthetic nanobodies (sybodies). Our results provide previously unknown insights into the molecular mechanism of a key transporter involved in bacterial pathogenesis and establish a basis for inhibition of the phosphocholine modification pathway across bacterial phyla.

Whole wheat flour has a shorter shelf life than refined wheat flour due to off-flavor development. An untargeted liquid chromatography/mass spectrometry (LC/MS) flavoromics approach was applied to identify compounds that negatively impact the flavor liking in whole wheat bread made from aged flours. The chemical profiles of thirteen breads made from aged flours were obtained using LC/MS and modeled by orthogonal partial least squares (OPLS) to predict flavor liking. Top predictive chemical features (negatively correlated) were identified as pinellic acid (9S,12S,13S-trihydroxy-10E-octadecenoic acid), 12,13-dihydroxy-9Z-octadecenoic acid, and 1-(9Z,12Z-octadecadienoyl)-sn-glycero-3-phosphocholine. The sensory analysis confirmed the three compounds increased the bitterness intensity of the bread samples. The formation of the trihydroxy fatty acid bitter compound, pinellic acid (9S,12S,13S-trihydroxy-10E-octadecenoic acid), was impacted by the lipoxygenase activity of the flour; however, there was no influence on the formation of 12,13-dihydroxy-9Z-octadecenoic acid or 1-(9Z,12Z-octadecadienoyl)-sn-glycero-3-phosphocholine. Additionally, the concentrations of all bitter compounds were significantly higher in bread made from aged flour versus non-aged flour.

The insula plays a critical role in many neuropsychological disorders. Research investigating its neurochemistry with magnetic resonance spectroscopy (MRS) has been limited compared with cortical regions. Here, we investigate the within-session and between-session reproducibility of metabolite measurements in the insula on a 3T scanner. We measure N-acetylaspartate + N-acetylaspartylglutamate (tNAA), creatine + phosphocreatine (tCr), glycerophosphocholine + phosphocholine (tCho), myo-inositol (Ins), glutamate + glutamine (Glx), and γ-aminobutyric acid (GABA) in one cohort using a j-edited MEGA-PRESS sequence. We measure tNAA, tCr, tCho, Ins, and Glx in another cohort with a standard short-TE PRESS sequence as a reference for the reproducibility metrics. All participants were scanned 4 times identically: 2 back-to-back scans each day, on 2 days. Preprocessing was done using LCModel and Gannet. Reproducibility was determined using Pearson’s r, intraclass-correlation coefficients (ICC), coefficients of variation (CV%), and Bland–Altman plots. A MEGA-PRESS protocol requiring averaged results over two 6:45-min scans yielded reproducible GABA measurements (CV% = 7.15%). This averaging also yielded reproducibility metrics comparable to those from PRESS for the other metabolites. Voxel placement inconsistencies did not affect reproducibility, and no sex differences were found. The data suggest that MEGA-PRESS can reliably measure standard metabolites and GABA in the insula.

High-resolution mass spectrometry is the foremost technique for qualitative and quantitative lipidomics analyses. Glycerophospholipids and sphingolipids, collectively termed polar lipids, are commonly investigated by hyphenated liquid chromatography-mass spectrometry (LC-MS) techniques that reduce aggregation effects and provide a greater dynamic range of detection sensitivity compared to shotgun lipidomics. However, automatic polar lipid identification is hindered by several isobaric and isomer mass overlaps, which cause software programs to often fail to correctly annotate the lipid species. In the present paper, a buffer modification workflow based on the use of labeled and unlabeled acetate ions in the chromatographic buffers was optimized by Box-Behnken design of the experiments and applied to the characterization of phosphocholine-containing lipids in human plasma samples. The contemporary generation of [M + CH3COO]-, [M + CD3COO]-, and [M - CH3]- coupled with a dedicated data processing workflow, which was specifically set up on Compound Discoverer software, allowed us to correctly determine adduct composition, molecular formulas, and grouping, as well as granting a lower false-positive rate and streamlining the manual validation step compared to commonly employed lipidomics platforms. The proposed workflow represents a robust yet easier alternative to the existing approaches for improving lipid annotation, as it does not require extensive sample pretreatment or prior isotopic enrichment or derivatization.

Simple carbohydrates are associated with the enhanced risk of cardiovascular disease and adverse changes in lipoproteins in the organism. Conversely, sugars are known to exert a stabilizing effect on biological membranes, and this effect is widely exploited in medicine and industry for cryopreservation of tissues and materials. In view of elucidating molecular mechanisms involved in the interaction of mono- and disaccharides with biomimetic lipid systems, we study the alteration of dielectric properties, the degree of hydration, and the rotational order parameter and dipole potential of lipid bilayers in the presence of sugars. Frequency-dependent deformation of cell-size unilamellar lipid vesicles in alternating electric fields and fast Fourier transform electrochemical impedance spectroscopy are applied to measure the specific capacitance of phosphatidylcholine lipid bilayers in sucrose, glucose and fructose aqueous solutions. Alteration of membrane specific capacitance is reported in sucrose solutions, while preservation of membrane dielectric properties is established in the presence of glucose and fructose. We address the effect of sugars on the hydration and the rotational order parameter for 1-palmitoyl-2-oleoyl-sn-glycero-3- phosphocholine (POPC) and 1-stearoyl-2-oleoyl-sn-glycero-3- phosphocholine (SOPC). An increased degree of lipid packing is reported in sucrose solutions. The obtained results provide evidence that some small carbohydrates are able to change membrane dielectric properties, structure, and order related to membrane homeostasis. The reported data are also relevant to future developments based on the response of lipid bilayers to external physical stimuli such as electric fields and temperature changes.

Sphingolipids are a class of lipids acting as key modulators of many physiological and pathophysiological processes. Hydroxylation patterns have a major influence on the biophysical properties of sphingolipids. In this work, we have studied the mechanism of action of hydroxylated lipids in sphingomyelin synthase (SMS). The structures of the two human isoforms, SMS1 and SMS2, have been generated through neural network supported homology. Furthermore, we have elucidated the reaction mechanism that allows SMS to recover the choline head from a phosphocholine (PC) and transfer it to ceramide, and we have clarified the role of the hydroxyl group in the interaction with the enzyme. Finally, the effect of partial inhibition of SMS on the levels of PC and sphingomyelin was calculated for different rate constants solving ordinary differential equation systems.

Sphingolipids are a structurally diverse class of lipids predominantly found in the plasma membrane of eukaryotic cells. These lipids can laterally segregate with other saturated lipids and cholesterol into lipid rafts; liquid-ordered (Lo) microdomains that act as organizing centers within biomembranes. Owing the vital role of sphingolipids for lipid segregation, controlling their lateral localization is of utmost significance. Hence, we made use of the light-induced trans-cis isomerization of azobenzene-modified acyl chains, to develop a set of photoswitchable sphingolipids, with different headgroups (hydroxyl, galactosyl, phosphocholine) and backbones (sphingosine, phytosphingosine, tetrahydropyran (THP)-blocked sphingosine), able to shuttle between liquid-ordered (Lo) and liquid-disordered (Ld) regions of model membranes upon irradiation with UV-A (λ = 365 nm) and blue (λ = 470 nm) light, respectively. Using combined high-speed atomic force microscopy, fluorescence microscopy, and force spectroscopy, we investigated how these active sphingolipids laterally remodel supported bilayers upon photo-isomerization, notably in terms of domain area changes, height mismatch, line tension, and membrane piercing. Hereby, we show that all sphingosine-(Azo-β-Gal-Cer, Azo-SM, Azo-Cer) and phytosphingosine-based (Azo-α-Gal-PhCer, Azo-PhCer) photolipids behave similarly, promoting a reduction in Lo domain area when in the UV-adapted cis-isoform. In contrast, azo-sphingolipids having THP groups that block H-bonding at the sphingosine backbone (Azo-THP-SM, Azo-THP-Cer) induce an increase in the Lo domain area when in cis, accompanied by a major rise in height mismatch and line tension. These changes were fully reversible upon blue light-triggered isomerization of the various lipids back to trans, pinpointing the role of interfacial interactions for the formation of stable Lo lipid raft domains.

Molekule 1,2-dipalmitoil-sn-glicero-3-fosfokolina (engl. 1,2-dipalmitoyl-sn-glicero-3- phosphatidylcholine, DPPC) u vodenom mediju stvaraju hidrirane dvosloje koji se, ovisno o temperaturi, nalaze u određenoj fazi. Prijelaz DPPC-a iz faze gela (Lβ') u fazu fluida (Lα), odnosno glavni fazni prijelaz opažen pri temperaturi mekšanja (Tm), prekinut je valovitom fazom (Pβ') koja se opaža pri temperaturi pretprijelaza (Tp). Budući da su Tm i Tp iznimno osjetljive na ugradnju organskih molekula u lipidni dvosloj, posebice onih koje su kadre primati i otpuštati proton, u sklopu ovog diplomskog rada istraženi su višestruki hidrirani dvosloji DPPC lipida u odsutnosti i prisutnosti male količine palmitinske kiseline (engl. palmitic acid, PA) u puferima različitih pH vrijednosti. Tm i Tp određene su analizom vibracijskih potpisa metilenskih (νsCH2) i fosfatnih (νasPO2−) skupina iz temperaturno-ovisnih FT-IR spektara. Dobiveni podaci su djelomično upotpunjeni onima dobivenima iz razlikovno-pretražne kalorimetrije (engl. differential scanning calorimetry, DSC). 1,2-dipalmitoyl-sn-glycero-3-phosphocoline form hydrated bilayers in an aqueous medium which, depending on the temperature, can be found in certain phase. The transition from gel (Lβ') to fluid phase (Lα), assigned as the main phase transition with a peak at melting point temperature (Tm), is interrupted by the ripple phase (Pβ') observed at the pretransition temeprature (Tp). Since Tm and Tp are extremely sensitive to the incorporation of organic molecules into the lipid bilayer, especially those capable of receiving and releasing a proton, multiple hydrated bilayers of DPPC lipids in the absence and presence of small amounts of palmitic acid (PA) in buffers of different pH values have been investigated across this thesis. Tm and Tp have been determined by the analysis of vibrational signatures of methylene (νsCH2) and phosphate (νasPO2−) groups from temperature-dependent FT-IR spectra. These data have been partially supplemented by those obtained from differential scanning calorimetry (DSC).

Objective: This study aimed to investigate the associations between dietary one-carbon metabolism-related nutrients (betaine, choline, methionine, folate, vitamin B6, and vitamin B12) and fluorosis among the Chinese population in an area known for coal-burning fluorosis.Methods: A cross-sectional study was conducted, with 653 fluorosis patients and 241 non-fluorosis participants. Dietary intake was acquired using a validated semi-quantitative 75-item food frequency questionnaire. The risk associations were assessed by unconditional logistical regression.Results: We observed a significant inverse association between dietary betaine, total choline, methionine, folate, vitamin B6, and choline species and fluorosis. The adjusted OR (95% CI) in the highest quartile of consumption compared with the lowest were 0.59 (0.37–0.94) (P-trend = 0.010) for betaine intake, 0.45 (0.28–0.73) (P-trend = 0.001) for total choline intake, 0.45 (0.28–0.72) (P-trend < 0.001) for methionine intake, 0.39 (0.24–0.63) (P-trend < 0.001) for folate intake, 0.38 (0.24–0.62) (P-trend < 0.001) for vitamin B6 intake, and 0.46 (0.28–0.75) (P-trend = 0.001) for total choline plus betaine intake. Dietary intakes of choline-containing compounds, phosphatidylcholine, free choline, glycerophosphocholine, and phosphocholine were also inversely associated with lower fluorosis (all P-trend < 0.05). No significant associations were observed between dietary vitamin B12 or sphingomyelin and fluorosis.Conclusion: The present study suggested that the higher dietary intakes of specific one-carbon metabolism-related nutrients, such as betaine, choline, methionine, folate, and vitamin B6, are associated with lower fluorosis prevalence.

The cytokine transforming growth factor-beta (TGF-beta) can induce normal breast epithelial cells to take on a mesenchymal phenotype, termed epithelial-to-mesenchymal transition (EMT). While the transcriptional and proteomic changes during TGF-beta-induced EMT have been described, the metabolic rewiring that occurs in epithelial cells undergoing EMT is not well understood. Here, we quantitively analyzed the TGF-beta-induced metabolic reprogramming during EMT of non-transformed NMuMG mouse mammary gland epithelial cells using nuclear magnetic resonance (NMR) spectroscopy. We found that TGF-beta elevates glycolytic and tricarboxylic acid (TCA)-cycle activity and increases glutaminolysis. Additionally, TGF-beta affects the hexosamine pathway, arginine-proline metabolism, the cellular redox state, and strongly affects choline metabolism during EMT. TGF-beta was found to induce phosphocholine production. A kinase inhibitor RSM-93A that inhibits choline kinase alpha (CHK alpha) mitigated TGF-beta-induced changes associated with EMT, i.e., increased filamentous (F)-actin stress fiber formation and N-Cadherin mesenchymal marker expression.

Many studies have confirmed the enzymatic activity of a mammalian phosphatidylcholine phospholipase C (PC-PLC), which produces diacylglycerol (DAG) and phosphocholine through the hydrolysis of phosphatidylcholine (PC) in the absence of ceramide. However, the protein(s) responsible for this activity have never yet been isolated. Based on the fact that tricyclodecan-9-yl-potassium xanthate (D609) can inhibit both PC-PLC and sphingomyelin synthase (SMS) activities, and SMS1 and SMS2 have a conserved catalytic domain which could mediates a nucleophilic attack on the phosphodiester bond of PC, we hypothesized that both SMS1 and SMS2 might have PC-PLC activity. In the current study, we found that purified recombinant SMS1 and SMS2 but not SMSr (sphingomyelin synthase related protein) have PC-PLC activity. Moreover, we prepared liver-specific Sms1/global Sms2 double knockout (dKO) mice. We found that liver PC-PLC activity was significantly reduced and steady state levels of PC and DAG in the liver were regulated by the deficiency, in comparison with control mice. Using adenovirus, we expressed Sms1 and Sms2 genes in the liver of the dKO mice, respectively, and found that expressed SMS1 and SMS2 can hydrolyze PC to produce DAG and phosphocholine. Thus, SMS1 and SMS2 exhibit PC-PLC activity in vitro and in vivo.

Phosphoethanolamine methyltransferase (PEAMT), a kind of S-adenosylmethionine-dependent methyltransferases, plays an essential role in many biological processes of plants, such as cell metabolism, stress response, and signal transduction. It is the key rate-limiting enzyme that catalyzes the three-step methylation of ethanolamine-phosphate (P-EA) to phosphocholine (P-Cho). To understand the unique function of PEAMT in soybean (Glycine max) lipid synthesis, we cloned two phosphoethanolamine methyltransferase genes GmPEAMT1 and GmPEAMT2, and performed functional identification. Both GmPEAMT1 and GmPEAMT2 contain two methyltransferase domains. GmPEAMT1 has the closest relationship with MtPEAMT2, and GmPEAMT2 has the closest relationship with CcPEAMT. GmPEAMT1 and GmPEAMT2 are located in the nucleus and endoplasmic reticulum. There are many light response elements and plant hormone response elements in the promoters of GmPEAMT1 and GmPEAMT2, indicating that they may be involved in plant stress response. The yeast cho2 opi3 mutant, co-expressing Arabidopsis thaliana phospholipid methyltransferase (PLMT) and GmPEAMT1 or GmPEAMT2, can restore normal growth, indicating that GmPEAMTs can catalyze the methylation of phosphoethanolamine to phosphate monomethylethanolamine. The heterologous expression of GmPEAMT1 and GmPEAMT2 can partially restore the short root phenotype of the Arabidopsis thaliana peamt1 mutant, suggesting GmPEAMTs have similar but different functions to AtPEAMT1.

Oxidized phospholipids have diverse biological activities, many of which can be pathological, yet how they are inactivatedin vivois not fully understood. Here we present evidence that a previously unsuspected host lipase, acyloxyacyl hydrolase (AOAH), can play a significant role in reducing the pro-inflammatory activities of two prominent products of phospholipid oxidation, 1-palmitoyl-2-glutaryl-sn-glycero-3- phosphocholine (PGPC) and 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC). AOAH removed the sn-2 and sn-1 acyl chains from both lipids and reduced their ability to induce macrophage inflammasome activation and cell deathin vitroand acute lung injuryin vivo. In addition to transforming Gram-negative bacterial lipopolysaccharide from stimulus to inhibitor, its most studied activity, AOAH can inactivate these important danger-associated molecular pattern (DAMP) molecules and reduce tissue inflammation and cell death.

Phosphorus (P) is an essential nutrient for plants. Membrane lipid remodeling is an adaptive mechanism for P-starved plants that replaces membrane phospholipids with non-P galactolipids, presumably to retrieve scarce P sources and maintain membrane integrity. Whereas metabolic pathways to convert phospholipids to galactolipids are well-established, the mechanism by which phospholipid biosynthesis is involved in this process remains elusive. Here, we report that phospho-base N-methyltransferases 1 and 2 (PMT1 and PMT2), which convert phosphoethanolamine to phosphocholine (PCho), are transcriptionally induced by P starvation. Shoots of seedlings of pmt1 pmt2 double mutant showed defective growth upon P starvation; however, membrane lipid profiles were unaffected. We found that P-starved pmt1 pmt2 with defective leaf growth had reduced PCho content, and the growth defect was rescued by exogenous supplementation of PCho. We propose that PMT1 and PMT2 are induced by P starvation to produce PCho mainly for leaf growth maintenance, rather than for phosphatidylcholine biosynthesis, in membrane lipid remodeling.

The nonshivering heat production in mitochondria-rich brown or beige adipocytes allows rodents and humans to adapt to cold stress (1). The best-characterized thermogenic effector is uncoupling protein 1 (UCP1), which dissipates energy as heat by proton transport across the mitochondrial inner membrane. Of note, there are also important UCP1-independent pathways mediating brown adipose tissue (BAT) thermogenesis (2). The discovery of significant brown and beige adipose depots in humans initiated efforts to leverage the thermogenic process as a means to increase the metabolic rate to prevent metabolic disease, such as obesity and diabetes (3). Currently, there are limited browning regimens for increased energy expenditure in humans short of cold exposure (4). In PNAS, Jiang et al. (5) investigated how the enzyme phosphoethanolamine and phosphocholine phosphatase 1 (PHOSPHO1) regulates thermogenesis in BAT, triggering changes in systemic insulin sensitivity and energy balance. PHOSPHO1 catalyzes the hydrolysis of phosphocholine (PC) to choline as well as of phosphoethanolamine (PEA) to ethanolamine and in the process, releases an inorganic phosphate group. PHOSPHO1 is not widely expressed, although it has been well characterized as the PC and PEA phosphatase in bone, where free inorganic phosphate is essential for bone mineralization (6). Additionally, PHOSPHO1 is a key regulator that coordinates the change in PC and phospholipid composition during terminal erythropoiesis (7). It is remarkable that bone and red blood cell progenitors rely on different aspects of PHOSPHO1 enzymatic activity. The authors discovered that PHOSPHO1 is highly enriched in murine BAT and accumulates during in vitro differentiation of primary brown adipocytes. PHOSPHO1 expression also increased during cold exposure in mice. Furthermore, they determined that metabolically active BAT from humans at the fetal stage has high levels of PHOSPHO1 vs. adult fat tissues. There is little known about the role of PHOSPHO1 in adipose tissue. Based on the literature, the … [↵][1]1To whom correspondence may be addressed. Email: philipp.scherer{at}utsouthwestern.edu. [1]: #xref-corresp-1-1

In Brazil, cutaneous leishmaniasis is caused predominantly by L. (V.) braziliensis. The few therapeutic drugs available exhibit several limitations, mainly related to drug toxicity and reduced efficacy in some regions. Miltefosine (MF), the only oral drug available for leishmaniasis treatment, is not widely available and has not yet been approved for human use in Brazil. Our group previously reported the existence of differential susceptibility among L. (V.) braziliensis clinical isolates. In this work, we further characterized three of these isolates of L. (V.) braziliensis chosen because they exhibited the lowest and the highest MF half maximal inhibitory concentrations and were therefore considered less tolerant or more tolerant, respectively. Uptake of MF, and also of phosphocholine, were found to be significantly different in more tolerant parasites compared to the less sensitive isolate, which raised the hypothesis of differences in the MF transport complex Miltefosine Transporter (MT)-Ros3. Although some polymorphisms in those genes were found, they did not correlate with the drug susceptibility phenotype. Drug efflux and compartmentalization were similar in the isolates tested, and amphotericin B susceptibility was retained in MF tolerant parasites, suggesting that increased fitness was also not the basis of observed differences. Transcriptomic analysis revealed that Ros3 mRNA levels were upregulated in the sensitive strain compared to the tolerant ones. Increased mRNA abundance in more tolerant isolates was validated by quantitative PCR. Our results suggest that differential gene expression of the MT transporter complex is the basis of the differential susceptibility in these unselected, naturally occurring parasites., Graphical abstract Image 1, Highlights • Brazilian L. (V.) braziliensis isolates vary in mitefosine susceptibility. • Diminished drug internalization was observed in more tolerant isolates. • Drug susceptibility did not correlate with SNPs in MT-Ros3 genes. • Drug efflux and compartmentalization were similar in the isolates tested. • Increased drug sensitivity is accompanied by Ros3 mRNA upregulation.

Resveratrol (RSV) is a well-known natural derivative with a wide range of biological and pharmacological activities. Despite of these demonstrated properties, it exhibits low both aqueous solubility and chemical stability and therefore low bioavailability. Consequently, the major concern of the technological research is to exploit delivery systems able to overcome bioavailability problems. In the recent past liposomes have been successfully studied for these purposes. In this paper, 1H-NMR spectroscopy, Nuclear Overhauser Spectroscopy (NOESY) as well as Paramagnetic Relaxation Enhancements (PRE) experiments have been carried out to quantitatively investigate the incorporation of resveratrol, at both the liposome preparation stage and by preformed liposomes, also with the aim to characterize resveratrol- soybean phosphatidylcholine (P90G) lipid bilayer interactions. Overall results of 1H NMR spectroscopy analysis suggest that RSV is located nearby the phosphocholine headgroups and also provide quantitative data on the incorporation of RSV (5% w/w), which corresponds to a 150-fold increase with respect to the solubility of RSV in water. Beside, considering that the same level of RSV incorporation was obtained via spontaneous uptake by preformed P90G liposomes, it can be concluded that RSV easily diffuses through the lipid bilayer., SCOPUS: ar.j, info:eu-repo/semantics/published

Deamidation of asparagine (Asn) residues of monoclonal antibodies (mAbs) plays a pivotal role in the in vivo/vitro degradation or efficacy loss of biopharmaceuticals. However, a major challenge for MS analysis of deamidation of Asn-containing peptides in mAbs, is due to the fact that there is only a 1 Da mass shift between the native form (Asn residues) and deamidated forms (n-aspartyl (n-Asp) and isoaspartyl (isoAsp) residues with identical mass). Therefore, a chromatographic separation of the deamidated proteins and/or the peptides derived therefrom is needed prior to MS analysis. In this study, the monolithic column with various stationary phases, including reverse phase (RP), single phospholipid-functionalized and mixed phospholipid-functionalized monoliths, were prepared for the separation of the deamidation-sensitive signature peptide (IYPTNGYTR) of trastuzumab and its two deamidated products, n-Asp55 residue IYPTDGYTR and isoAsp55 residue IYPTisoDGYTR. Compared to the RP monolith, the phospholipid-functionalized monoliths provided mixed-mode interactions and exhibited better peak shape and separation selectivity. The effect of the parameters, including the type and concentration of buffer, temperature and pH value on the separation performance were investigated. Under the optimal conditions, the three peptides were fully separated on a mixed phosphocholine (PC) / phosphatidic acid (PA) functionalized monolith (poly (MDPC60PA40-co-EDMA)) due to the joint contribution of hydrophobic and electrostatic interactions. Therefore, the novel method based on the mixed phospholipids-functionalized monolithic column exhibited good potential for the analysis of deamidated peptides, which will be useful for the in-depth study of post-translational modifications of mAbs.

Effects of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) on the oxidative stability were determined in soybean oil–water system at different locations including at the interface of air–oil, in the middle of oil, and at the interface of oil–water. Also, profile changes of tocopherols were determined during UV irradiation for 18 days. Although no significant changes in tocopherol profiles were observed at three different locations irrespective of DOPC from 0 to 1250 μmol/kg oil, addition of DOPC increased total tocopherols, α-tocopherol, and δ-tocopherol whereas content of β + γ tocopherols did not increase at any locations. Moisture content in water–oil interface was higher than other locations while those were not consistent at different DOPC concentration. Added DOPC significantly decreased oxidative stability from 250 to 830 μmol/kg oil compared to controls (p